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Differential Effects of IFN-β on the Survival and Growth of Human Vascular Smooth Muscle and Endothelial Cells.

Sano E, Tashiro S, Tsumoto K, Ueda T - Biores Open Access (2015)

Bottom Line: To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC).The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells.In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo , Chiba, Japan .

ABSTRACT
It has been documented that interferon (IFN)-β is effective against the genesis of atherosclerosis or hyperplastic arterial disease in animal model. The main mechanism of the efficacy was antiproliferative action on the growth of vascular smooth muscle cells (SMC). To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC). IFN-β enhanced SMC apoptosis in serum starved medium. Conversely, EC apoptosis induced by serum and growth factor deprivation was inhibited by IFN-β. The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities. Anti-apoptotic bcl-2 mRNA was also up-regulated in EC. IFN-β inhibited SMC growth in a dose dependent manner. However, the growth of EC was rather enhanced by a low dose of IFNs. The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells. The growth stimulation on EC was considered to link with increase of S and G2/M phase cells. SMC produced IFN-β in response to various stimulants. However, IFN-β was not induced in EC. These suggested that endogenous IFN-β from SMC may act on EC and affect to EC functions. In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth. These effects were considered to contribute to a cure against hyperplastic arterial diseases as the mechanisms in the efficacy of IFN-β.

No MeSH data available.


Related in: MedlinePlus

P53 status and function of HCASMC. (A) PCR–single-strand conformation polymorphism (PCR-SSCP) analysis of p53 gene. The cells were serially subcultured in the medium with or without 20 ng/mL platelet derived growth factor (PDGF)-BB. After preparation of genomic DNA from these cells, PCR and SSCP analysis were performed. PCR products of each exon were electrophoresed and visualized using fluorescent imaging analyzer. N, normal control; M, mutated control; S1, cell sample without PDGF-BB; S2, cell sample with PDGF-BB. (B) Growth suppressor function of p53. The cells of 1×104 were seeded in 24 well plates. After the preculture for 24 h, the cells were proliferated in 2% fetal calf serum (FCS) supplemented medium together with two different p53 antisense (AS1, AS2) and control oligodeoxynucleotide. The proliferated cells were counted after 6 days using coulter counter. Average cell number in four wells and SE of the mean were demonstrated. **p<0.01, compared with control and p53 antisense addition. (C) Effects of IFNs on the p53 expression (Western blotting analysis). HCASMC were cultured until 80% confluent and treated with 1,000 IU/mL of IFN-α, -β, and -γ. The harvested cells were lysed with RIPA buffer and the extracts from the cells were analyzed for p53 contents. The proportion of band intensity normalized by these of β-actin is shown in the table.
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f2: P53 status and function of HCASMC. (A) PCR–single-strand conformation polymorphism (PCR-SSCP) analysis of p53 gene. The cells were serially subcultured in the medium with or without 20 ng/mL platelet derived growth factor (PDGF)-BB. After preparation of genomic DNA from these cells, PCR and SSCP analysis were performed. PCR products of each exon were electrophoresed and visualized using fluorescent imaging analyzer. N, normal control; M, mutated control; S1, cell sample without PDGF-BB; S2, cell sample with PDGF-BB. (B) Growth suppressor function of p53. The cells of 1×104 were seeded in 24 well plates. After the preculture for 24 h, the cells were proliferated in 2% fetal calf serum (FCS) supplemented medium together with two different p53 antisense (AS1, AS2) and control oligodeoxynucleotide. The proliferated cells were counted after 6 days using coulter counter. Average cell number in four wells and SE of the mean were demonstrated. **p<0.01, compared with control and p53 antisense addition. (C) Effects of IFNs on the p53 expression (Western blotting analysis). HCASMC were cultured until 80% confluent and treated with 1,000 IU/mL of IFN-α, -β, and -γ. The harvested cells were lysed with RIPA buffer and the extracts from the cells were analyzed for p53 contents. The proportion of band intensity normalized by these of β-actin is shown in the table.

Mentions: The p53 gene status of HCASMC was examined using PCR-SSCP method to clarify the effects of IFN-β on the expression of p53 related genes since it has been documented that advanced vascular SMC growth occurs by p53 gene mutation. HCASMC were serially cultured for over a month in the presence or absence of PDGF-BB. After the preparation of genomic DNA, the mutations in exon 5, 6, 7, and 8 were analyzed by rhodamine labeled primers of these exons. PCR products were electrophoresed and visualized using fluorescent imaging analyzer. As shown in Fig. 2A, any obvious alterations in these exons were not found indicating the p53 is wild type. The growth suppressor function of the p53 was examined using two different p53 antisense oligodeoxynucleotides. The cells were cultured together with antisense and control nucleotides for 6 days. As shown in Fig. 2B, the growth of the cells was stimulated by the addition of p53 antisense nucleotides indicating the p53 of HCASMC possesses growth suppressor function. Next, the effects of IFNs on the p53 activation were examined by western blotting analysis. The cells were treated with 1,000 IU/mL of various IFNs. As shown in Fig. 2C, the expression of p53 were downregulated by these IFNs with time elapse. This suggested the activation of p53 by IFNs although the phospholylated p53 could not be detected by used anti-phospholylated p53 monoclonal antibody. The p53 protein level was most downregulated by IFN-β.


Differential Effects of IFN-β on the Survival and Growth of Human Vascular Smooth Muscle and Endothelial Cells.

Sano E, Tashiro S, Tsumoto K, Ueda T - Biores Open Access (2015)

P53 status and function of HCASMC. (A) PCR–single-strand conformation polymorphism (PCR-SSCP) analysis of p53 gene. The cells were serially subcultured in the medium with or without 20 ng/mL platelet derived growth factor (PDGF)-BB. After preparation of genomic DNA from these cells, PCR and SSCP analysis were performed. PCR products of each exon were electrophoresed and visualized using fluorescent imaging analyzer. N, normal control; M, mutated control; S1, cell sample without PDGF-BB; S2, cell sample with PDGF-BB. (B) Growth suppressor function of p53. The cells of 1×104 were seeded in 24 well plates. After the preculture for 24 h, the cells were proliferated in 2% fetal calf serum (FCS) supplemented medium together with two different p53 antisense (AS1, AS2) and control oligodeoxynucleotide. The proliferated cells were counted after 6 days using coulter counter. Average cell number in four wells and SE of the mean were demonstrated. **p<0.01, compared with control and p53 antisense addition. (C) Effects of IFNs on the p53 expression (Western blotting analysis). HCASMC were cultured until 80% confluent and treated with 1,000 IU/mL of IFN-α, -β, and -γ. The harvested cells were lysed with RIPA buffer and the extracts from the cells were analyzed for p53 contents. The proportion of band intensity normalized by these of β-actin is shown in the table.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4497630&req=5

f2: P53 status and function of HCASMC. (A) PCR–single-strand conformation polymorphism (PCR-SSCP) analysis of p53 gene. The cells were serially subcultured in the medium with or without 20 ng/mL platelet derived growth factor (PDGF)-BB. After preparation of genomic DNA from these cells, PCR and SSCP analysis were performed. PCR products of each exon were electrophoresed and visualized using fluorescent imaging analyzer. N, normal control; M, mutated control; S1, cell sample without PDGF-BB; S2, cell sample with PDGF-BB. (B) Growth suppressor function of p53. The cells of 1×104 were seeded in 24 well plates. After the preculture for 24 h, the cells were proliferated in 2% fetal calf serum (FCS) supplemented medium together with two different p53 antisense (AS1, AS2) and control oligodeoxynucleotide. The proliferated cells were counted after 6 days using coulter counter. Average cell number in four wells and SE of the mean were demonstrated. **p<0.01, compared with control and p53 antisense addition. (C) Effects of IFNs on the p53 expression (Western blotting analysis). HCASMC were cultured until 80% confluent and treated with 1,000 IU/mL of IFN-α, -β, and -γ. The harvested cells were lysed with RIPA buffer and the extracts from the cells were analyzed for p53 contents. The proportion of band intensity normalized by these of β-actin is shown in the table.
Mentions: The p53 gene status of HCASMC was examined using PCR-SSCP method to clarify the effects of IFN-β on the expression of p53 related genes since it has been documented that advanced vascular SMC growth occurs by p53 gene mutation. HCASMC were serially cultured for over a month in the presence or absence of PDGF-BB. After the preparation of genomic DNA, the mutations in exon 5, 6, 7, and 8 were analyzed by rhodamine labeled primers of these exons. PCR products were electrophoresed and visualized using fluorescent imaging analyzer. As shown in Fig. 2A, any obvious alterations in these exons were not found indicating the p53 is wild type. The growth suppressor function of the p53 was examined using two different p53 antisense oligodeoxynucleotides. The cells were cultured together with antisense and control nucleotides for 6 days. As shown in Fig. 2B, the growth of the cells was stimulated by the addition of p53 antisense nucleotides indicating the p53 of HCASMC possesses growth suppressor function. Next, the effects of IFNs on the p53 activation were examined by western blotting analysis. The cells were treated with 1,000 IU/mL of various IFNs. As shown in Fig. 2C, the expression of p53 were downregulated by these IFNs with time elapse. This suggested the activation of p53 by IFNs although the phospholylated p53 could not be detected by used anti-phospholylated p53 monoclonal antibody. The p53 protein level was most downregulated by IFN-β.

Bottom Line: To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC).The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells.In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo , Chiba, Japan .

ABSTRACT
It has been documented that interferon (IFN)-β is effective against the genesis of atherosclerosis or hyperplastic arterial disease in animal model. The main mechanism of the efficacy was antiproliferative action on the growth of vascular smooth muscle cells (SMC). To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC). IFN-β enhanced SMC apoptosis in serum starved medium. Conversely, EC apoptosis induced by serum and growth factor deprivation was inhibited by IFN-β. The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities. Anti-apoptotic bcl-2 mRNA was also up-regulated in EC. IFN-β inhibited SMC growth in a dose dependent manner. However, the growth of EC was rather enhanced by a low dose of IFNs. The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells. The growth stimulation on EC was considered to link with increase of S and G2/M phase cells. SMC produced IFN-β in response to various stimulants. However, IFN-β was not induced in EC. These suggested that endogenous IFN-β from SMC may act on EC and affect to EC functions. In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth. These effects were considered to contribute to a cure against hyperplastic arterial diseases as the mechanisms in the efficacy of IFN-β.

No MeSH data available.


Related in: MedlinePlus